Fig. 6: Redistribution of p53 binding underlies genetic reprogramming and cancer phenotypes.

a Top motifs enriched in lost (MCF10A-unique) DARs within TADs of up- or down-regulated genes. Background group is gained DARs. b RNA levels of TP53 in MCF10A (blue) and G12V (red) cells determined by RNA-seq. Data points and average (bar graph) are presented. p-value determined by DESeq2. c Immunostaining of p53 (top panel) in MCF10A and G12V cells. Bar = 20μm. p53 protein levels in MCF10A and G12V cells as measured by western blot analysis (bottom). Representative results of at least two biological repeats are shown. d Variation in RNA levels |log2FC| following 4 h Nutlin-3a treatment in MCF10A cells. Gray—all expressed genes, red –up-regulated genes after HRas transformation, blue—down-regulated genes after HRas transformation and green—down-regulated genes that have lost regulatory sites with a p53 motif in their domain. *p < 0.001, Wilcox test. e % of gained (red) and lost (blue) DARs overlapping p53 binding sites from MCF10A and G12V ChIP-seq in up and down TADs. *p < 0.001, proportional test. f Example for a lost DAR that overlaps with lost p53 binding site (pink box). Blackline indicates p53 motif. Chromosomal coordinates in Mb of human hg19 genome build are presented. g Heatmap displaying k-means clustering of p53 ChIP-Seq data in the two cell types. The ATAC-seq data were arranged to match the order of loci found by clustering p53 ChIP-Seq. Four kb around the ChIP-seq peaks are displayed. h Cell proliferation with 5 μM Nutlin-3a was measured by XTT assay. The relative number of cells, compared to day 0 is presented for MCF10A (blue line) and G12V cells (red line). *p = 0.0002 T-test (i, j) Representative images showing the migration capability and growth pattern of MCF10A and G12V cells with or without Nutlin-3a. Scale bar (i) =400 µm, Scale bar (j) =100 µm.